Convergence and divergence of the photoregulation of pigmentation and cellular morphology in Fremyella diplosiphon

Abstract

Photosynthetic pigment accumulation and cellular and filament morphology are regulated reversibly by green light (GL) and red light (RL) in the cyanobacterium Fremyella diplosiphon during complementary chromatic adaptation (CCA). The photoreceptor RcaE (regulator of chromatic adaptation), which appears to function as a light-responsive sensor kinase, controls both of these responses. Recent findings indicate that downstream of RcaE, the signaling pathways leading to light-dependent changes in morphology or pigment synthesis and/or accumulation branch, and utilize distinct molecular components. We recently reported that the regulation of the accumulation of the GL-absorbing photosynthetic accessory protein phycoerythrin (PE) and photoregulation of cellular morphology are largely independent, as many mutants with severe PE accumulation defects do not have major disruptions in the regulation of cellular morphology. Furthermore, morphology can be disrupted under GL without impacting GL-dependent PE accumulation. Most recently, however, we determined that the disruption of the cpeR gene, which encodes a protein that is known to function as an activator of PE synthesis under GL, results in disruption of cellular morphology under GL and RL. Thus, apart from RcaE, CpeR is only the second known regulator to impact morphology under both light conditions in F. diplosiphon.

Introduction

Many photosynthetic organisms are sessile, i.e., they have very limited abilities to move within their environments. This sessile nature dictates that such organisms adapt their patterns of growth and development to fluctuations in parameters of their external environment to survive and maximize productivity. Autotrophic organisms that harvest sunlight and convert it to chemical energy during photosynthesis possess mechanisms and adaptive responses to fluctuating environmental conditions, particularly light. The impact of variations in light on photosynthetic organisms, including photosynthetic bacteria and plants, has been investigated widely.

Photomorphogenesis describes light-dependent changes in growth, development and cellular metabolism that are largely associated with increased fitness in organisms. The freshwater cyanobacterium Fremyella diplosiphon (also designated Calothrix sp PCC 7601) has been used as a model cyanobacterium for exploring the photomorphogenic response commonly known as complementary chromatic adaptation (CCA). CCA is a light-dependent acclimation process during which chromatically-adapting organisms such as F. diplosiphon alter the pigment composition of light-harvesting complexes named phycobilisomes (PBSs) that are associated physically and functionally with the photosynthetic photosystems.^1,2 During CCA, other organismal properties, including cellular morphology and filament length, also change in response to variations in the prevalent wavelengths of light in the external photoenvironment.^1,2

The fine tuning of the pigment composition of the PBSs that occurs during CCA results in an increase in light absorption, which is associated with enhanced photosynthetic capacity under varying light conditions.³ This process, in addition to the photoreversible morphological changes, i.e., changes in cell shape and filament length,^1,4 is primarily responsive to red light (RL) and green light (GL) in F. diplosiphon.^1,2 The same photoreceptor RcaE (regulator of chromatic adaptation) that is involved in the regulation of light-dependent pigmentation in F. diplosiphon^5,6 also controls the photoreversible morphological changes.^4,7 Our analyses of the factors that function downstream of RcaE in the regulation of distinct aspects of CCA, i.e., pigmentation vs. morphology, indicate that some common factors are involved, particularly under RL;⁷ yet, a number of distinct components contribute to light-dependent control of pigmentation and/or morphology in this organism.^8-11 Recent results provide an emerging picture that downstream of the central RL- and GL-responsive photoreceptor RcaE the regulation of morphology and pigmentation, the principal features of CCA, are mediated by independently driven processes at the molecular level.

Dissecting the regulatory mechanisms controlling distinct aspects of CCA in F. diplosiphon

Insight into the molecular pathways that control distinct aspects of CCA, namely the photoregulation of pigmentation vs. cellular and/or filament morphology, has only recently begun to emerge.^4,6-11 Characterization of a ΔcpcF mutant, which lacks virtually all PBPs, provided strong evidence that cellular morphology is regulated reversibly by GL and RL, largely separate from the impacts of these light qualities on PBP pigment accumulation.¹⁰ A ΔcpcF mutant accumulates less than 5% of phycoerythrin (PE) and phycocyanin (PC) levels of WT and lower allophycocyanin (AP) levels than WT, yet the length of ΔcpcF cells under GL and RL differed little from WT.¹⁰ We have assessed several PE mutants to determine whether regulation of PE accumulation under GL is linked directly to the photoregulation of morphology in F. diplosiphon.^9,11 We isolated mutants harboring mutations in several PE structural genes, including apoprotein-encoding genes cpeBA,⁹ linker gene cpeD,⁹ and linker gene cpeE,¹¹ all of which exhibit minor GL-dependent cellular morphology defects though they exhibit major PE deficiencies under GL. These mutants also have lower levels of PC and AP,^9,11 which may be reflective of cellular adaptation; i.e., as there is little to no PE available for GL absorption, the cells may degrade or reduce synthesis of other phycobiliproteins that are not able to directly absorb GL. Thus, in these PE structural mutants, the energy deficit associated with PE deficiency could be the cause of an indirect, yet minor, effect of PE deficiency on cellular length in GL. Furthermore, in support of molecular independence of the regulation of morphology and pigmentation, we isolated a mutant with a mutation in a still unidentified gene that severely impacts PE accumulation under GL, yet the mutant shows no alteration in cellular morphology under this condition.¹¹

Rca regulatory proteins have been shown to be required for photoregulation of cellular morphology in F. diplosiphon. RcaE is required under both GL and RL,⁴ and RcaF and RcaC are required under RL.⁷ However, the impact of these proteins on morphology appears to occur largely independent of their impact on pigmentation, particularly under GL for RcaC and RcaF.^4,7 This observation that pigmentation and morphology are regulated largely independently is also supported in part by the fact that WT cells show signs of light-dependent changes in morphology before changes in pigmentation are detected.⁴ Apart from Rca proteins, TonB has also been implicated in GL-dependent regulation of cell width and operates independent of RcaE function (Fig. 1).⁸ TonB has no apparent impact on pigmentation.⁸ The molecular mechanism(s) employed by TonB to impact cell width is still under investigation.

Figure 1.

Model for regulation of pigmentation and cellular morphology in Fremyella diplosiphon. Regulatory photoreceptor RcaE responds to both green light (GL) and red light (RL) and regulates a phosphorelay cascade, including two response regulators, RcaF and DNA-binding transcriptional activator RcaC. Under GL, RcaE is proposed to maintain RcaF in an unphosphorylated state, resulting in unphosphorylated RcaC, which in turn upregulates the expression of the cpeCDESTR operon encoding linker components of phycoerythrin (PE) and PE regulatory activator cpeR. CpeR, in turn, upregulates PE apoprotein-encoding operon cpeBA and the pebAB operon encoding phycoerythrobilin chromophore biosynthetic enzymes. These GL-dependent changes result in PE synthesis and accumulation. Under RL, the Rca system is proposed to function ultimately through RcaE-dependent phosphorylation of RcaC (i.e., RcaC-P) to repress expression of PE-related operons and upregulate expression of the cpcB2A2 operon encoding phycocyanin (PC) components, pcyA encoding a phycocyanobilin chromophore biosynthetic enzyme, and cpcF as a part of the cpcEF operon encoding a dimeric PC lyase. These RL-associated changes result in PC accumulation. Cellular morphology changes also occur in response to changes in GL and RL, which are mediated in part by RcaE. Both RcaE and CpeR are required for apposite morphology regulation under GL, conditions resulting in longer brick-shaped cells, and RL, conditions associated with smaller, round cells. RcaF and RcaC are also required to attain the RL-dependent cellular morphology; whereas TonB is required specifically under GL.

Our most recent studies support a role for regulator CpeR in the light-dependent control of cellular morphology under both RL and GL,⁹ whereas its impact on PE pigment accumulation is GL-specific.^12,13 Notably, CpeR accumulates under both RL and GL, though its expression is higher under GL in both the SF33 and UTEX 481 strains of F. diplosiphon (Fig. 2). CpeR has also been shown to impact cellular functions under RL, as the accumulation of light-dependent protochlorophyllide oxidoreductase (por) gene transcript is higher under RL in a ΔrcaE/ΔcpeR double mutant than in either WT or a ΔrcaE single mutant.¹⁴ This observation suggests that under RL, CpeR functions to repress accumulation of the por transcript in F. diplosiphon. Nostoc punctiforme has two copies of cpeR, one that is GL-induced and one that is constitutively present in both GL and RL.¹⁵ This finding suggests that in some systems, apart from F. diplosiphon, CpeR proteins also function in RL and GL. Markedly, F. diplosiphon significantly represses PE accumulation under RL, but PE is not absolutely absent.² This observation, together with our cpeR transcript analysis (Fig. 2), indicates that active expression of PE-related genes, including cpeR, occurs under RL, though the expression levels are greatly upregulated by GL exposure.

Figure 2.

RT-PCR analysis of the expression of cpeR. RT-PCR amplification of cpeR in SF33 wild-type pigmentation strain and UTEX 481 wild-type (UTEX) strain grown under green light (GL) or red light (RL). RT-PCR was conducted essentially as described previously,⁹ with the following modifications: RT-PCR was performed using primer sets designed for cpeR, i.e., forward primer 5′-CCTCCAGAAGCACAGAAG-3′ and reverse primer 5′-TTGTGGCAGGGTGTATGT-3′, and the gene-specific annealing temperature for cpeR was 49°C, which resulted in a product 239 bp in length. The transcript level of the 23S ribosomal gene (ribo) was used as a control for each sample. The no reverse transcriptase (NRT) enzyme control is shown.

Previously, CpeR has been shown to be epistatic to, i.e., functions downstream of, RcaC in regards to PE accumulation (Fig. 1; see ref.12). The morphology of ΔrcaC cells differs from SF33 parental cells most significantly under RL, but is quite similar to SF33 under GL.⁷ Thus, CpeR may be epistatic to RcaC under RL for the regulation of cellular morphology. However, whether this relationship also holds under GL, conditions under which the cellular morphology of a ΔcpeR mutant differs significantly from UTEX 481 WT,⁹ whereas a ΔrcaC mutant is quite similar to parental SF33,⁷ will require additional investigation. To further delineate or elucidate the specific role(s) of CpeR under RL vs. GL and determine its relationship to RcaC in the photoregulation of cellular morphology, epistasis studies would be conducted most appropriately in the same parental background, as the reason(s) that different effects are seen with the same mutations in the UTEX 481 wild type (WT) vs. SF33 WT pigmentation background require additional investigation.

The signaling pathways controlling synthesis and accumulation of PE and regulation of cellular morphology share upstream factors, e.g., RcaE,⁴ RcaF,^4,7 RcaC^4,7 and CpeR,⁹ but have distinct regulatory components as well. Distinct morphology components include GL-dependent factors downstream of RcaE,⁷ RL-dependent CpeR function,⁹ TonB function in GL,⁸ others for which the identity of the mutation has not been elucidated,¹¹ and likely others yet that have not been identified at all. Components that appear to largely impact pigmentation are CpcF under RL and GL,¹⁰ and CpeD,⁹ CpeE,¹¹ and CpeBA⁹ under GL.

Ongoing efforts to dissect the mechanisms of regulation of distinct aspects of CCA in F. diplosiphon

There are a number of additional outstanding questions regarding the photoregulation of cellular morphology in F. diplosiphon. These questions include the following: (1) What is the physiological relevance of morphology being regulated by light? (2) What additional factors function downstream of RcaE in the regulation of morphology under GL? (3) What photoreceptor regulates GL-dependent upregulation of tonB expression, which impacts morphology apart from RcaE function? (4) To what does RcaC signal to modulate/control RL-dependent morphology? With regard to the first question, readers are referred to a more comprehensive review of recent insights into environmental and developmental regulation of cellular morphology in cyanobacteria and associated fitness benefits to the organisms (see ref.16). However, all of these outstanding questions will require additional experimentation to result in the definitive identification and characterization of the molecular nature and biochemical function(s) of the effectors involved in discrete aspects of CCA.

Glossary

Abbreviations:

AP

allophycocyanin

CCA

complementary chromatic adaptation

chla

chlorophyll a

GL

green light

PBP

phycobiliprotein

PBS

phycobilisomes

PC

phycocyanin

PE

phycoerythrin

rca

regulation of chromatic adaptation

RL

red light

WT

wild type

Whitaker MJ, Pattanaik B, Montgomery BL. Characterization of Green Mutants in Fremyella diplosiphon Provides Insight into the Impact of Phycoerythrin Deficiency and Linker Function on Complementary Chromatic Adaptation. Biochem Biophys Res Commun. 2011;404:52–6. doi: 10.1016/j.bbrc.2010.11.056.

Pattanaik B, Whitaker MJ, Montgomery BL. Regulation of Phycoerythrin Synthesis and Cellular Morphology in Fremyella diplosiphon Green Mutants. Biochem Biophys Res Commun. 2011;413:182–8. doi: 10.1016/j.bbrc.2011.08.051.